Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson–Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C → T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.     

The C-terminal Lysine of Ogg2 DNA Glycosylases is a Major Molecular Determinant for Guanine/8-Oxoguanine Distinction

7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.     

Clostridium acetobutylicum 8-oxoguanine DNA glycosylase (Ogg) differs from eukaryotic Oggs with respect to opposite base discrimination

During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine-DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine-DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG.C relative to 8-oxoG.A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair.     

Specificity of stimulation of human 8-oxoguanine-DNA glycosylase by AP endonuclease

Human 8-oxoguanine-DNA glycosylase OGG1 is an enzyme that removes abundant oxidative lesion 8-oxoguanine (8-oxoG) from DNA. Excision of 8-oxoG by OGG1 is inhibited by the abasic DNA reaction product and is stimulated by AP endonuclease APEX1. Besides 8-oxoG, OGG1 shows activity towards several other base lesions. Here we report that APEX1 efficiently stimulates OGG1 on good substrates (8-oxoadenine, 8-oxoinosine, or 6-methoxy-8-oxoguanine opposite to cytosine) but the stimulation is low or absent with poor OGG1 substrates (8-oxoadenine or 8-oxoinosine opposite to thymine; 8-oxoG or 8-aminoguanine opposite to adenine; 8-oxonebularine, 8-metoxyguanine, inosine or guanine opposite to cytosine). APEX1 significantly improves the ability of OGG1 to excise 8-aminoguanine from its naturally occurring pair with cytosine, making it possible that OGG1 repairs this lesion. Overall, APEX1 serves to improve specificity of OGG1 for its biologically relevant substrates.     

Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats

Background

Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration.

Methods

We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats.

Results

It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions.

Conclusions

Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats.

General significance

The findings are important for validation of the OXYS rat strain as a model of mammalian aging.     

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.     

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.     

Functional Characterization of 8-Oxoguanine DNA Glycosylase of Trypanosoma cruzi

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-)/(-) (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2)O(2)). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2)O(2) of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.     

The capacity to remove 8-oxoG is enhanced in newborn neural stem/progenitor cells and decreases in juvenile mice and upon cell differentiation

In mammalian cells, 8-oxoguanine DNA glycosylase-1 (OGG1) is the main DNA glycosylase for the removal of 8-oxoguanine (8-oxoG). 8-oxoG, one of the most common products of the oxidative attack of DNA, is a premutagenic lesion that accumulates spontaneously at high frequencies in the genome. In this study, Ogg1 mRNA expression was detected throughout embryonic development in mice. In situ hybridization showed that in the neonatal brain, Ogg1 expression was detected in a distinct layer of cells in the medial wall of the lateral ventricle, which may correspond to ependymal cells, and in some scattered cells in the subventricular zone (SVZ), a brain region rich in neural stem/progenitor cells. Using neurospheres as a model for the study of neural stem/progenitor cells, we found that both the expression and activity of Ogg1 were high in neurospheres derived from newborn mice and decreased in adults and upon induction of cell differentiation. Furthermore, Ogg1 was shown to be the major DNA glycosylase initiating 8-oxoG repair in neurospheres. Our results strongly indicate that enhanced DNA repair capacity is an important mechanism by which neural stem/progenitor cells maintain their genome.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana

Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsis thaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate characteristic of all bifunctional DNA glycosylases/lyases. Consistent with its in vitro activity, expression of AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxoG repair. Our results suggest that AtOgg1 is an structural and functional homologue of Ogg1 and establish the presence of two distinct 8-oxoG repair enzymes in Arabidopsis.     

Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.     

Comparative analysis of 8-oxoG:C, 8-oxoG:A,A:C and C:C DNA repair in extracts from wild type or 8-oxoG DNA glycosylase deficient mammalian and bacterial cells

We have investigated repair of DNA containing 8-oxoguanine and certain mismatches in cell-free extracts from mouse embryonic fibroblasts (MEFs) using a plasmid substrate with a single lesion at a defined position. Repair synthesis was monitored in a small restriction fragment with different size single strands in order to follow the fate of repair reactions in both strands at the same time. An important part of the study was to assess the role of OGG1 in various repair reactions and the experiments were carried out with extracts from mouse embryonic fibroblasts diploid for a mogg1 deletion (Ogg1(-/-)) as well as wild type. In wild type, DNA containing 8-oxoG:C was repaired in the expected fashion predominantly through short-patch repair. Overall repair was reduced to 20% in the Ogg1(-/-) extracts and to 40% if only long-patch repair was considered. The 8-oxoG:A pair was processed similarly in wild type and Ogg1(-/-) extracts and repair synthesis at A as well as at 8-oxoG could be demonstrated, however, to the same extent in Ogg1(-/-) and wild type for both strands. Extracts from Ogg1(-/-) behaved normally in the correction of A:C and C:C mismatches, with a strong bias for correction of A for A:C and no significant strand discrimination for C:C. Similar experiments with extracts from Escherichia coli showed a 50% reduction in the repair of 8-oxoG:C in fpg extracts and an increase to 50% above wild type in mutY. These results show that the mouse OGG1 is the major enzyme for 8-oxoG repair in the MEF cells and does not participate in mismatch repair of A:C or C:C. Furthermore, 8-oxoG opposite A appears to be repaired by a two-step repair pathway with sequential removal of A and 8-oxoG mediated by enzymes different from OGG1.     

极端嗜热古菌8oxoG DNA糖苷酶的研究进展

7,8二氢-8-氧鸟嘌呤(7,8-dihydro-8-oxoguanine,8oxoG)是一种常见的DNA损伤碱基。由于8oxoG能够与腺嘌呤配对,在DNA中的8oxoG被修复之前进行复制,DNA将会产生GC→TA的突变,从而造成基因组的不稳定。目前,碱基切除修复(Base excision repair,BER)是修复DNA中8oxoG的经典途径,其中8oxoGDNA糖苷酶(8-oxoguanineDNAglycosylases,OGG)是启动BER途径的关键酶。研究发现,OGG能够识别和切除DNA中的8oxoG,从而阻止细胞内GC→TA突变的积累。目前,OGG分为3个家族:OGG1、OGG2和AGOG (archaeal 8oxoG DNA glycosylase),广泛分布于细菌、古菌和真核生物。古菌基因组的序列表明,它们至少编码一种OGG。目前,对源自细菌和真核生物的OGG已进行了大量的研究,但是关于极端嗜热古菌OGG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌OGG的研究进展,并对今后的研究提出了展望。     

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H₂O₂ or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.     

Repair activities of 8-oxoguanine DNA glycosylase from Archaeoglobus fulgidus, a hyperthermophilic archaeon.

Oxidative DNA damage is caused by reactive oxygen species formed in cells as by products of aerobic metabolism or of oxidative stress. The 8-oxoguanine (8-oxoG) DNA glycosylase from Archaeoglobus fulgidus (Afogg), which excises an oxidatively-damaged form of guanine, was overproduced in Escherichia coli, purified and characterized. A. fulgidus is a sulfate-reducing archaeon, which grows at between 60 and 95 degrees C, with an optimum growth at 83 degrees C. The Afogg enzyme has both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities, with the latter proceeding through a Schiff base intermediate. As expected for a protein from a hyperthermophilic organism, the enzyme activity is optimal near pH 8.5 and 60 degrees C, denaturing at 80 degrees C, and is thermally stable at high levels of salt (500mM). The Afogg protein efficiently cleaves oligomers containing 8-oxoG:C and 8-oxoG:G base pairs, and is less effective on oligomers containing 8-oxoG:T and 8-oxoG:A mispairs. While the catalytic action mechanism of Afogg protein is likely similar to the human Ogg1 (hOgg1), the DNA recognition mechanism and the basis for 8-oxoG substrate specificity of Afogg differ from that of hOgg.